Summary of work: The long-term goals are to identify and characterize key components of the intrinsic genetic program that controls development and function of male germ cells. The approaches being used are to identify genes expressed specifically in male germ cells, use transgenic mice and in vitro assays to identify promoter elements and transcription factors regulating their expression, and apply the gene knockout approach to define the roles of the proteins they encode. Many genes are expressed only in male germ cells and we are focusing on a few encoding proteins whose functions can be assayed and are likely to be important in gamete development or function. We isolated the mouse and human GAPDS genes for a key isozyme in the glycolytic pathway, glyceraldehyde 3-phosphate dehydrogenase, which is expressed only in male germ cells and believed to have a key role in regulating generation of ATP required for fertilization. Antibodies prepared to synthetic peptides localized GAPDS to the fibrous sheath of the sperm flagellum in mouse and human. Yeast two-hybrid screens identified a WW-domain protein (FBP3) which binds to a proline-rich region of mouse GAPDS. We are mapping the binding domains on each protein and testing the hypothesis that FBP3 anchors GAPDS to the fibrous sheath. In addition, molecular modeling studies indicate that residues surrounding the substrate-binding pocket may account for differences in the effects of inhibitors on the somatic and germ cell isozymes. Recombinant GAPDS is being prepared and used to study the interaction of GAPDS with its natural substrate, natural cofactor, and reproductive toxicants. The toxicants include (S)-3-chlorolactaldehyde, a metabolite of the industrial solvent epichlorohydrin, and ornidizole, used therapeutically as an anti-microbial agent, that appear to act as competitive inhibitors of substrate binding to GAPDS. We have also used gene targeting to test the hypothesis that sperm from Gapds knockout mice will be unable to produce the ATP necessary to achieve hyperactivated motility and will be unable to fertilize eggs. Gapds +/- mice have been produced and Gapds -/- mice will be available in the near future for study. We demonstrated that GAPDS binds to the fibrous sheath, a cytoskeletal structure in the sperm flagellum. The major structural protein of the fibrous sheath is a protein kinase A (PKA) anchoring protein (AKAP) encoded by the AKAP4 gene. We used yeast two-hybrid assays to identify proteins that associate with AKAP4. We found that PKA binds to AKAP4 and may regulate cAMP-dependent protein-phosphorylation essential for activation of sperm motility. Yeast two-hybrid assays, alanine and valine scanning-mutagenesis, and pull-down assays were used to define the amino acids of AKAP4 responsible for the binding of regulatory (R) subunits of the PKA tetramer. RI alpha-specific and dual RI alpha/RII alpha-specific binding motifs were identified. It was found that hydrophobic amino acids at three consensus positions on AKAPs are required for PKA binding and that specificity of PKA binding is determined by the size of the aliphatic side-chain on the amino acid in the middle position. This was verified by introducing point mutations into these motifs to switch between RI alpha-specific, RII alpha-specific, and RI alpha/RII alpha dual-specific binding. These findings represent an important advance in understanding the relationship between the primary sequence and the three-dimensional spatial distribution of residues within the amphipathic alpha-helix of AKAP anchoring domains that determine PKA binding specificity. They are also significant for the understanding of molecular mechanisms involved in PKA subtype localization within cells. We used yeast two-hybrid screens to identify additional proteins that bind to AKAP4 in the fibrous sheath. We found previously that male germ cell-specific glutathione S-transferase (GSTM5) and alternate forms of hexokinase 1 (HK1S) are present in the fibrous sheath. The yeast two-hybrid screens determined that AKAP3 and two novel proteins expressed only in spermatogenic cells also bind to AKAP4. In addition, Cre/loxP-mediated gene mutation was used to produce Akap4 gene knockout mice and the males were found to be infertile due to disruption of flagellar structure and function. Transgenic mice are being generated that express AKAP4 with point mutations that abolish RI alpha and RII alpha binding or that result in switches in PKA isoform binding. The transgenes are under the control of a promoter expressed only in spermatogenic cells. These animal models will allow in vivo studies of the function of AKAP4 and of the role of different PKA isoforms in sperm function, as well as the analysis of the interactions between AKAPs and PKAs. We also used gene targeting to disrupt expression of the PR1 and PR2 genes encoding protamines 1 and 2. These are highly basic nuclear proteins that replace histones following meiosis. They are thought essential for DNA compaction in spermatids, whose nuclei are haploid and lack nucleosomes. Spermatids are connected by cytoplasmic bridges, through which they share mRNA and protein. Disruption of one copy of either PR1 or PR2 led to altered sperm structure and function and failure to transmit the mutant allele through the germ line of male chimeras. We found that protein sharing leads to reduction in amount of protein by one-half in all spermatids, resulting in defective nuclear compaction during spermiogenesis. This appears to be the first observation of haplo-insufficiency leading to disruption of genetic inheritance in mammals. Significance: We have shown that male fertility is susceptible to disruption by spontaneous or induced mutations in genes expressed only in male germ cells and encoding proteins essential for the development and function of male gametes (e.g., protamines 1 and 2). In addition, such proteins may be targets for environmental chemicals that impair their function and cause male infertility without effects on somatic cells (e.g., GAPDS). Although many unique genes are expressed only in male germ cells, few have been shown previously to encode proteins that are critical for spermatogenesis. These studies are identifying and characterizing proteins required for germ cell development and function that are potential targets of male reproductive toxicants or may allow the development of highly specific male contraceptives.